Biomedical subjects

S Ståhl

Publications and source records attributed to S Ståhl.

At least 19 recordsLinked to original sources

PubMed2004

Selection and characterization of HER2/neu-binding affibody ligands.

Affibody (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

Amino Acid Sequence↗

PubMed2001

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Amino Acid Sequence↗

PubMed2001

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain.

The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.

Cell Wall↗

PubMed2001

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex.

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.

Amino Acid Sequence↗

PubMed2001

Tachykinin NK(2) receptor mediates contraction and ion transport in rat colon by different mechanisms.

We have characterized the tachykinin NK(2) receptor-mediated contraction and vectorial ion transport responses in the muscularis mucosae and mucosa of the rat isolated distal colon, respectively. The tachykinin NK(2) receptor-selective antagonist nepadutant (c([(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta))) produced competitive antagonism of [betaAla(8)]neurokinin A-(4-10)-induced contraction (pK(B) = 9.3) in the muscularis mucosae, and insurmountable blockade of increases in short-circuit current (I(sc)) responses (pK(B) = 8.6) in the mucosa. However, this latter effect was completely reversed by washout of the antagonist. [betaAla(8)]Neurokinin A-(4-10)-induced contractions were unaffected by indomethacin (3 microM). In sharp contrast, I(sc) responses induced by [betaAla(8)]neurokinin A-(4-10) (100 nM) were inhibited (>70%) by indomethacin (3 microM), while I(sc) responses to substance P (3 microM) were unchanged. Our study provides the first evidence that in the same organ stimulation of tachykinin NK(2) receptors leads to two independent responses mediated by different effector mechanisms both of which are blocked (albeit with different kinetics) by the potent and selective tachykinin NK(2) receptor antagonist, nepadutant.

Animals↗

PubMed2001

Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column.

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Animals↗

PubMed2001

Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains.

Ni(2+)-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni(2+)-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni(2+)-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

Amino Acid Sequence↗

PubMed2001

Surface display on gram positive bacteria.

Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, "second generation" vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Gram-positive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.

Animals↗

PubMed2000

High-throughput protein expression of cDNA products as a tool in functional genomics.

A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.

Amino Acid Sequence↗

PubMed2000

Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation.

We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

Adjuvants, Immunologic↗

PubMed2000

Expression profile viewer (ExProView): a software tool for transcriptome analysis.

A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a "virtual chip," in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.

Data Display↗

PubMed2000

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein.

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.

Bioreactors↗

PubMed2000

Staphylococcal surface display and its applications.

Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

Animals↗

PubMed2000

Design and production of recombinant subunit vaccines.

The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases. The use of recombinant-DNA techniques has facilitated the development of new principles for design and production of subunit vaccines. First of all, the properties of a target protein immunogen can be improved by the use of gene-fusion technology or by the creation of specific changes, to generate 'second-generation protein vaccines'. Properties that can be modified include protein solubility, protein stability, in vivo half-lives, etc. In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites. The recombinant subunit vaccine can furthermore be adapted by gene-fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems. Also in passive vaccination strategies, i.e. the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important. Humanized antibodies and antibody fusion proteins represent common present anti-infectious-disease agents. The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.

Adjuvants, Immunologic↗

PubMed2000

The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme.

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis. Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tektl staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.

Animals↗

PubMed2000

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen.

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.

Animals↗

PubMed2000

Staphylococcal surface display of metal-binding polyhistidyl peptides.

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

Cadmium↗

PubMed1999

Engineering of a Staphylococcus carnosus surface display system by substitution or deletion of a Staphylococcus hyicus lipase propeptide.

Surface display of recombinant proteins on bacteria and phages has become an important topic in bioscience. A system for the display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signal and propeptide from a Staphylococcus hyicus lipase for translocation and since the propeptide is of considerable size (207 amino acids) and not processed in S. carnosus, we have investigated the possibility to delete or substitute the propeptide for smaller protein domains, to thereby improve the surface display system. A set of new vectors was constructed and the surface expression of model proteins was investigated by various methods, including fluorescence-activated cell sorting. The results suggest that the propeptide region indeed can be deleted when proteins which are easily secretable are displayed. In contrast, the propeptide seems to be advantageous for translocation of inefficiently secreted proteins. Moreover, our study also presents a rational strategy for how to monitor the engineering efforts for the optimization of a surface display system.

Base Sequence↗